Journal: Oncotarget

Article Title: MED15 overexpression in prostate cancer arises during androgen deprivation therapy via PI3K/mTOR signaling

doi: 10.18632/oncotarget.13860

Figure Lengend Snippet: a. Reduced MED15 expression in LNCaP cell which were treated with MED15 specific siRNA shown by western blot. b. MTT assay reveals 50% reduction of LNCaP cell viability after MED15 knockdown when cells were grown under androgen deprivation for 72 hours. Absorbance at 595nm was normalized to control cells (one-sample t-test, *** p < 0.001). Data are represented as mean ± SEM. c. Percentage of Annexin V-positive LNCaP cells which were treated with scrambled or MED15 specific siRNA under androgen deprivation (independent t-test, ** p < 0.01). Data are represented as mean ± SEM. d. Reduced AR expression in MED15 knockdown cells compared to control cells shown by western blot. Similar MED1 levels in control and MED15 knockdown cells.

Article Snippet: ICC was performed according to published protocol for cultured cell lines by Cell Signaling Technology (Danvers, MA) using an anti-MED15 rabbit polyclonal antibody (1:200, 11566-1-AP, Proteintech, Chicago, IL) and anti-MED1 mouse monoclonal antibody (1:100, H-7, Santa Cruz, Texas, US).

Techniques: Expressing, Western Blot, MTT Assay, Knockdown, Control

Journal: Nature Structural & Molecular Biology

Article Title: Rational optimization of a transcription factor activation domain inhibitor

doi: 10.1038/s41594-023-01159-5

Figure Lengend Snippet: a , Predicted structure of AR, colored by structure-prediction confidence from high (blue) to low (yellow). The domains and the native NLS are highlighted. b , Live-cell STED imaging of representative ( n > 3) HEK293T cells transfected with AR constructs tagged with mEGFP. Cells were imaged after treatment with 10 nM DHT for 4 h. Scale bar, 5 μm. The dashed line indicates the nuclear periphery. c , Intensity of AR AD NMR resonances at different concentrations, relative to the intensity at 25 μM. The positions of Tau-1, Tau-5 and 23 FQNLF 27 are highlighted. Green circles indicate residues that were not visible (NV) or not assigned (NA), including residues in polyglutamine (pQ), polyproline (pP) and polyglycine (pG) tracts. Yellow and orange circles represent the positions of tyrosine residues substituted by serines in 8YtoS and 14YtoS; all tyrosine residues were substituted in 22YtoS. d , Fluorescence microscopy images of 40 µM AR-AD droplets (WT* and mutants) at 1 M NaCl. Scale bar, 10 μm. e , Scheme of the phase diagram of the AR AD and of how T c measurements at different solution conditions allow the phase separation capacity of the mutants to be ranked. f , T c measurements of AR AD (WT* and the tyrosine to serine mutants), as mean ± s.d. of three independent samples, at two different solution conditions. g , Representative merged confocal images of 15 µM MED1-IDR and 5 µM RNAPII-CTD droplets at 20 mM NaCl or 50 mM NaCl, respectively, and 10% ficoll before and after addition of 1 µM AR AD (WT* or 22YtoS). Scale bars, 5 μm. h , Quantification of AR AD partitioning in MED1-IDR (top) and RNAPII-CTD droplets (bottom), by measuring AR AD fluorescence intensity ( I (AR AD)). Boxes show the mean and the quartiles of all droplets, represented as colored dots from three replicated images. arb.u., arbitrary units. i , Representative ( n > 3) merged confocal images of MED1-IDR and RNAPII-CTD droplets obtained in 125 mM NaCl and 10% ficoll with and without the addition of 1 µM AR AD (WT* or 22YtoS). Scale bar, 5 μm. j , Normalized intensity plot of cross-sections from the images shown in i .

Article Snippet: Primary antibodies to the following proteins were added and incubated overnight at 4 °C: androgen receptor (ER179(2)) (Abacam, no. ab108341, 1:200), Nup153 (Abacam, QE5, no. ab24700, 1:200), Med1 (Abacam, no. ab64965, 1:200) and ARID1a/BAF250A (Cell Signalling, no. 12354, 1/200).

Techniques: Imaging, Transfection, Construct, Fluorescence, Microscopy

Journal: Nature Structural & Molecular Biology

Article Title: Rational optimization of a transcription factor activation domain inhibitor

doi: 10.1038/s41594-023-01159-5

Figure Lengend Snippet: a , Schematic of the method for small-molecule treatment of cells and the BioID experiment. b, BioID–MS of LNCaP MTID-AR-WT cells treated with EPI-001 (10 μM, 1 h), followed by treatment with DHT and biotin (2 h). Intensity data were obtained from SAINTq analysis. Shown is the log (FC) of the intensity of the interaction in inhibitor-treated versus DMSO-treated cells. Decreased interactors in inhibitor-treated cells relative to DMSO-treated cells are shown in blue; P < 0.05. The dashed lines mark where log (FC) < −1.5. Increased interactors in inhibitor-treated cells relative to DMSO-treated cells are shown in red; P < 0.05. The dashed lines mark where log (FC) > 1.5. Proteins of interest are annotated ( n = 3). c , As in b , BioID–MS of LNCaP MTID-AR-WT cells. Cells were treated for 1 h with 1ae (5 μM) and then for 2 h with DHT and biotin ( n = 3). d , (Total mean) TMean intensities of peptides identified by MS ( n = 3) from SAINTq data of individual proteins (bait), total interactors (all) or collated known AR interactors sourced from BioGrid ( https://thebiogrid.org/ ) were compared between LNCaP MTID-AR-WT cells treated with DMSO and those treated with small-molecule inhibitors. e , GO search terms of key biological processes and molecular functions in SAINTq intensity data from b and c (BFDR < 0.02, depleted = log (FC) < −1.5), obtained from the LNCaP MTID-AR-WT cells treated with small-molecule inhibitors versus DMSO. Full categories are available in Supplementary Data Table . f , BioID–MS of the LNCaP MTID-AR-WT interaction with Mediator complex in cells treated with small-molecule inhibitors versus DMSO. SAINTq data, color indicates strength of interaction change from logFC 10 TMean of intensity. g , The TMean intensity of interactions with the MED1 subunit of Mediator was compared between LNCaP MTID-AR-WT cells treated with DMSO or small-molecule inhibitors. SAINTq data were used. Statistical significance was determined by Student’s t -test against the control group. h , Results from a PLA in LNCaP MTID-AR-WT cells, using the indicated antibodies shown in cyan with DAPI staining shown in magenta. Cells were treated with small-molecule inhibitors or DMSO and DHT at the indicated times. Scale bars, 10 μm . i , Distributions of PLA foci per cell, each dot corresponds to a cell and boxes shown the mean and quartiles of all cells ( n > 20 cells).

Article Snippet: Primary antibodies to the following proteins were added and incubated overnight at 4 °C: androgen receptor (ER179(2)) (Abacam, no. ab108341, 1:200), Nup153 (Abacam, QE5, no. ab24700, 1:200), Med1 (Abacam, no. ab64965, 1:200) and ARID1a/BAF250A (Cell Signalling, no. 12354, 1/200).

Techniques: Control, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Med1 in LPS‐EC‐EVs serves as an IL‐21 activating TF. (a) Venn diagram of upregulated proteins in LPS‐EC‐EVs and IL‐21 predicted TFs showing that only Med1 was accumulated. (b) Immunoblotting combined with Ponceau S staining confirmed that Med1 was selectively enriched in LPS‐EC‐EVs rather than EC‐EVs ( n = 3). (c) Representative IEM image showing the presence of Med1 in LPS‐EC‐EVs. Scale bar, 100 nm. (d) 3D view indicated the GFP‐labelled EVs in the Treg nucleus (blue) after coculturing. (e) Immunoblotting analysis confirmed that Med1 was transferred into the Treg nucleus through EVs. Tregs cocultured with Flag‐Med1 LPS‐EC‐EVs and proceeded to isolate nuclear and cytoplasmic fractions ( n = 3). (f) Schematic model of the primer targeting sites (left) and Flag‐tagged ChIP‐qPCR analysis (right) demonstrated attachment of Med1 to the predicted region on IL‐21 ( n = 5). (g) Gel‐based imaging of Flag‐tagged ChIP‐qPCR products from (f). (h) Dual‐luciferase assays were performed to confirm the interaction between Med1 and IL‐21 ( n = 6). *** p < 0.005, **** p < 0.001. See also Figures and .

Article Snippet: For IEM, EVs were dried and permeabilized on nickel grids, then incubated with Med1 rabbit mAb (A700‐037‐T, Bethyl Laboratories, USA) at 4°C.

Techniques: Western Blot, Staining, ChIP-qPCR, Imaging, Luciferase

Journal:

Article Title:

doi:

Figure Lengend Snippet: Med1 in LPS‐EC‐EVs is the key modulator for Treg restriction. (a) Immunoblotting showed the Med1 expression in WT and Med1 knockdown ECs ( n = 3). (b) Med1 depletion efficiency in EVs from WT and Med1 knockdown ECs ( n = 3). FCM (c) and quantification (d) of CD25 + Foxp3 + double staining in Tregs exposed to Med1‐KD LPS‐EC‐EVs ( n = 3). (e) RT‐qPCR (left) and ELISA (right) of IL‐21 expression in Tregs and cell culture supernatants ( n = 3). (f) Seven‐day survival analysis of LPS‐IT mice in shMed1‐AAV‐LungX group and shNC‐AAV‐LungX group. Immunoblotting (g) and quantification (h) of Med 1 expression in BALF‐EVs after AAV‐shNC or AAV‐shMed1 infection ( n = 5). (i) Quantification of Tregs in BALF and BLNs from different group mice via FCM at 72 h after LPS administration ( n = 5). (j) Representative HE stained tissue sections and lung injury score evaluation ( n = 5 in each group). Scale bar, 100 µm. (k) Immunofluorescence staining and quantitative analysis of MPO ( n = 5 each group). Scale bar, 100 µm. (l and m) Immunohistochemical staining and quantitative analysis of Ly6G (l) and NE (m) ( n = 5 each group). Scale bar, 100 µm. * p <0.05, ** p < 0.01, *** p <0.005, **** p < 0.001. See also Figure .

Article Snippet: For IEM, EVs were dried and permeabilized on nickel grids, then incubated with Med1 rabbit mAb (A700‐037‐T, Bethyl Laboratories, USA) at 4°C.

Techniques: Western Blot, Expressing, Knockdown, Double Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Infection, Staining, Immunofluorescence, Immunohistochemical staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: Elevated Med1 expression in BALF‐EVs from ARDS patients. (a) Comparison of CD31 + EVs percentage in the BALF from non‐ARDS ( n = 5) and ARDS ( n = 9) patients. Immunoblotting (b) and quantification (c) of Med1 expression in BALF‐EVs from non‐ARDS ( n = 5) and ARDS patients ( n = 9). (d) IL‐21 concentrations in the BALF from non‐ARDS ( n = 5) and ARDS ( n = 9) patients. (e) Correlation analysis of different variables including CD31 + EV proportion, Med1 relative expression in BALF‐EVs and IL‐21 concentration in BALF. * p < 0.05, ** p < 0.01, **** p < 0.001. See also Figure .

Article Snippet: For IEM, EVs were dried and permeabilized on nickel grids, then incubated with Med1 rabbit mAb (A700‐037‐T, Bethyl Laboratories, USA) at 4°C.

Techniques: Expressing, Comparison, Western Blot, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Endothelium‐Treg Communication Through Extracellular Vesicle Transfer Exacerbates Acute Respiratory Distress Syndrome

doi: 10.1002/jev2.70235

Figure Lengend Snippet: IL‐21 signalling blockade ameliorates the EV‐induced Treg suppression. aIL‐21 (a), aIL‐21R (b) and Stattic (c) were seen to rescue EV‐induced Treg suppression in a dose‐dependent manner ( n = 3). (d) Immunoblotting of the phosphor‐STAT3 and STAT3 levels of Tregs exposed to LPS‐EC‐EVs after treatment with IL‐21 signalling inhibitors for 5 days ( n = 3). (e and f) Splenic naïve CD4 + T cells were isolated from WT and Il21 −/− mice and then polarized to Tregs with LPS‐EC‐EVs for 5 days. The percentage of Tregs were analyzed via FCM (e) and data were statistically analyzed (f), ( n = 6). (g) Schematic diagram of ALI induction in chimeras (created using Biorender.com). (h) Quantification of Tregs in BALF and BLNs from different chimeras via FCM ( n = 5). (i) Representative HE stained tissue sections and lung injury score evaluation ( n = 5 in each group). Scale bar, 100 µm. (j) Immunofluorescence staining and quantitative analysis of MPO ( n = 5 in each group). Scale bar, 100 µm. (k and l) Immunohistochemical staining and quantitative analysis of Ly6G (k) and NE (l) ( n = 5 in each group). Scale bar, 100 µm. ** p < 0.01, *** p < 0.005, **** p < 0.001. See also Figure .

Article Snippet: The following antibodies were used for the immunoblotting experiments: phosphor‐smad2 rabbit mAb (18338, CST, USA); smad2 rabbit mAb (5339, CST, USA); phosphor‐smad3 rabbit mAb (9520, CST, USA); smad3 rabbit mAb (9523, CST, USA); phosphor‐STAT5 rabbit mAb (25656, CST, USA); STAT5 rabbit mAb (94205, CST, USA); phosphor‐STAT3 rabbit mAb (9145, CST, USA); STAT3 rabbit mAb (9139, CST, USA); Foxp3 rabbit mAb (ab215206, Abcam, USA); Med1 rabbit mAb (A700‐037‐T, Bethyl Laboratories, USA); Flag tag rabbit mAb (14793, CST, USA); α‐tubulin rabbit pAb (11224‐1‐AP, proteintech, China); histone‐H3 rabbit pAb (17168‐1‐AP, proteintech, China); GM130 rabbit mAb (70767, CST, USA); Alix rabbit mAb (ab235377, Abcam, USA); CD63 rabbit pAb (ab315108, Abcam, USA); CD9 rabbit mAb (ab307085, Abcam, USA); β‐actin rabbit pAb (HPR conjugated) (ZF2001, ZFdows Bio, China) and HPR‐linked anti‐rabbit Ab (7074, CST, USA).

Techniques: Western Blot, Isolation, Staining, Immunofluorescence, Immunohistochemical staining